Multiple sclerosis (MS) is a chronic disease of the central nervous system, affecting upwards of 2 million people worldwide. Though traditionally considered an inflammatory disease, recent evidence has brought neurodegeneration to the spotlight, suggesting that axonal damage is crucial to the development of irreversible disability. Studies show that axonal degeneration occurs throughout the entire MS disease course. While the specific mechanisms causing axonal damage may differ at various stages, mitochondrial failure seems to be a common underlying theme. In an effort to elucidate the pathways leading to mitochondrial dysfunction and consequent axonal degeneration, the proposed experiments investigate potential effectors of mitochondrial permeability transition (PT). Mitochondrial PT occurs when a transient pore forms in the inner mitochondrial membrane leading to the influx of solutes, mitochondrial swelling, membrane rupture, release of pro-apoptotic molecules, and eventual cell death. Evidence points to the mitochondrial matrix protein cyclophilin D (CyPD) as a key regulator of PT. In addition, elevated reactive oxygen species (ROS) have been shown to induce PT and the resulting cascade of events. In this proposal, the effects of CyPD and p66ShcA (p66), a recently discovered mitochondrial redox enzyme and a potential upstream PT inducer, will be investigated in the context of mitochondrial dysfunction, neuronal survival, and axonal degeneration. Specifically, animals in which p66 and/or CyPD have been eliminated will be induced with the commonly used MS disease model experimental autoimmune encephalomyelitis (EAE), and the amount of axonal protection will be compared. In addition, in vitro neuronal cultures prepared from p66 and/or CyPD knockout mice will be treated with various agents implicated in MS neurodegeneration (e.g. inflammatory cytokines, nitric oxide, glutamate, ROS). Neuronal robustness, ROS production, and mitochondrial morphology changes will be assessed in the different genotypic cultures following treatment. Overall, these proposed studies will produce novel data that may bring us closer to understanding how ROS production, PT regulation, and mitochondrial dysfunction are involved in MS neurodegenerative pathogenesis. Ultimately, my overarching research goal is to contribute to the development of MS treatments that target axonal degeneration. In the past, MS has largely been considered a chronic inflammatory and demyelinating disease, driving most of the research and treatment development towards targeting the immune system. These anti-inflammatory agents, while marginally successful in reducing acute inflammatory lesions and clinical relapses, are largely ineffective in treating the irreversible disability of progressive MS. Increasing evidence shows that neurodegeneration impacts all stages of MS, and therefore, development of neuroprotective treatments is critical. Better understanding of the roles that p66 and CyPD play in PT, mitochondrial dysfunction, and axonal degeneration, as proposed in this research plan, will help define therapeutic strategies specifically directed at MS neurodegeneration. PUBLIC HEALTH RELEVANCE: Multiple sclerosis (MS) is a chronic debilitating disease that affects more than two million people worldwide. Currently, the treatments available provide only temporary symptom relief, and are ineffective in preventing the irreversible disability that afflicts MS patients. The research proposed in this training plan is designed to understand what causes this irreversible disability with the goal of defining new drug targets for which effective treatments can be developed.